DNA-binding and chromatin localization properties of CHD1.

CHD1 is a novel DNA-binding protein that contains both a chromatin organization modifier (chromo) domain and a helicase/ATPase domain. We show here that CHD1 preferentially binds to relatively long A.T tracts in double-stranded DNA via minor-groove interactions. Several CHD1-binding sites were found in a well-characterized nuclear-matrix attachment region, which is located adjacent to the intronic enhancer of the kappa immunoglobulin gene. The DNA-binding activity of CHD1 was localized to a 229-amino-acid segment in the C-terminal portion of the protein, which contains sequence motifs that have previously been implicated in the minor-groove binding of other proteins. We also demonstrate that CHD1 is a constituent of bulk chromatin and that it can be extracted from nuclei with 0.6 M NaCl or with 2 mM EDTA after mild digestion with micrococcal nuclease. In contrast to another chromo-domain protein, HP1, CHD1 is not preferentially located in condensed centromeric heterochromatin, even though centromeric DNA is highly enriched in (A+T)-rich tracts. Most interestingly, CHD1 is released into the cytoplasm when cells enter mitosis and is reincorporated into chromatin during telophase-cytokinesis. These observations lend credence to the idea that CHD1, like other proteins with chromo or helicase/ATPase domains, plays an important role in the determination of chromatin architecture.     

1H, 13C and 15N resonance assignments of an N-terminal domain of CHD4

Chromatin-remodeling proteins have a pivotal role in normal cell function and development, catalyzing conformational changes in DNA that ultimately result in changes in gene expression patterns. Chromodomain helicase DNA-binding protein 4 (CHD4), the defining subunit of the nucleosome remodeling and deacetylase (NuRD) complex, is a nucleosome-remodeling protein of the SNF2/ISWI2 family, members of which contain two chromo domains and an ATP-dependent helicase module. CHD3, CHD4 and CHD5 also contain two contiguous PHD domains and have an extended N-terminal region that has not previously been characterized. We have identified a stable domain in the N-terminal region of CHD4 and report here the backbone and side chain resonance assignments for this domain at pH 7.5 and 25 °C (BMRB No. 18906).     

A Structural Model for the DEAD Box Helicase YxiN in Solution: Localization of the RNA Binding Domain

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core.YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.     

The Tumor Suppressor Chromodomain Helicase DNA-binding Protein 5 (CHD5) Remodels Nucleosomes by Unwrapping

Although mutations or deletions of chromodomain helicase DNA-binding protein 5 (CHD5) have been linked to cancer and implicate CHD5 in tumor suppression, the ATP-dependent activity of CHD5 is currently unknown. In this study, we discovered that CHD5 is a chromatin remodeling factor with a unique enzymatic activity. CHD5 can expose nucleosomal DNA at one or two discrete positions in the nucleosome. The exposure of the nucleosomal DNA by CHD5 is dependent on ATP hydrolysis, but continued ATP hydrolysis is not required to maintain the nucleosomes in their remodeled state. The activity of CHD5 is distinct from other related chromatin remodeling ATPases, such as ACF and BRG1, and does not lead to complete disruption or destabilization of the nucleosome. Rather, CHD5 likely initiates remodeling in a manner similar to that of other remodeling factors but does not significantly reposition the nucleosome. While the related factor CHD4 shows strong ATPase activity, it does not unwrap nucleosomes as efficiently as CHD5. Our findings add to the growing evidence that chromatin remodeling ATPases have diverse roles in modulating chromatin structure.     

Human CHD2 Is a Chromatin Assembly ATPase Regulated by Its Chromo- and DNA-binding Domains

Chromodomain helicase DNA-binding protein 2 (CHD2) is an ATPase and a member of the SNF2-like family of helicase-related enzymes. Although deletions of CHD2 have been linked to developmental defects in mice and epileptic disorders in humans, little is known about its biochemical and cellular activities. In this study, we investigate the ATP-dependent activity of CHD2 and show that CHD2 catalyzes the assembly of chromatin into periodic arrays. We also show that the N-terminal region of CHD2, which contains tandem chromodomains, serves an auto-inhibitory role in both the DNA-binding and ATPase activities of CHD2. While loss of the N-terminal region leads to enhanced chromatin-stimulated ATPase activity, the N-terminal region is required for ATP-dependent chromatin remodeling by CHD2. In contrast, the C-terminal region, which contains a putative DNA-binding domain, selectively senses double-stranded DNA of at least 40 base pairs in length and enhances the ATPase and chromatin remodeling activities of CHD2. Our study shows that the accessory domains of CHD2 play central roles in both regulating the ATPase domain and conferring selectivity to chromatin substrates.     

The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex.

The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing. The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif. Prior to this study little was known about functional roles for these two domains. We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing. Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing. In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity. Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex. Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p. Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro. The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these. Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix. Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.     

Solution structure of the BRK domains from CHD7

CHD7 is a member of the chromodomain helicase DNA binding domain (CHD) family of ATP-dependent chromatin remodelling enzymes. It is mutated in CHARGE syndrome, a multiple congenital anomaly condition. CHD7 is one of a subset of CHD proteins, unique to metazoans that contain the BRK domain, a protein module also found in the Brahma/BRG1 family of helicases. We describe here the NMR solution structure of the two BRK domains of CHD7. Each domain has a compact betabetaalphabeta fold. The second domain has a C-terminal extension consisting of two additional helices. The structure differs from those of other domains present in chromatin-associated proteins.     

Interaction of the bacteriophage T4 gene 59 helicase loading protein and gene 41 helicase with each other and with fork, flap, and cruciform DNA

Bacteriophage T4 gene 59 helicase loading protein accelerates the loading of T4 gene 41 DNA helicase and is required for recombination-dependent DNA replication late in T4 phage infection. The crystal structure of 59 protein revealed a two-domain alpha-helical protein, whose N-terminal domain has strong structural similarity to the DNA binding domain of high mobility group family proteins (Mueser, T. C., Jones, C. E., Nossal, N. G., and Hyde, C. C. (2000) J. Mol. Biol. 296, 597-612). We have previously shown that 59 protein binds preferentially to fork DNA. Here we show that 59 protein binds to completely duplex forks but cannot load the helicase unless there is a single-stranded gap of more than 5 nucleotides on the fork arm corresponding to the lagging strand template. Consistent with the roles of these proteins in recombination, we find that 59 protein binds to and stimulates 41 helicase activity on Holliday junction DNA, and on a substrate that resembles a strand invasion structure. 59 protein forms a stable complex with wild type 41 helicase and fork DNA in the presence of adenosine 5'-O-(thiotriphosphate). The unwinding activity of 41 helicase missing 20 C-terminal amino acids is not stimulated by 59 protein, and it does not form a complex with 59 protein on fork DNA.     

Nonstructural protein 5B of hepatitis C virus

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, a-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.     

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins

BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.     

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.     

Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller.